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Figure 3-9 The separation of the 20 hr cytotoxicity assay for the NK cell- mediated lysis of CMV-infected fibroblasts into distinct activation and effector phases

PBMCs were co-cultured for 16 hr in a transwell system prior to their use as effector cells in a 4 hr cytotoxicity assay. Fibroblasts were either left uninfected, or were infected with CMV strain AD 169 for 4 days at an MOI of 2. The cells were then radiolabelled for use as target cells in the cytotoxicity assay, or added to co-cultures as follows. PBMCs were added to the upper transwell chamber, separated by a semi-porous membrane from the lower chamber, which contained medium alone (CONTROL), uninfected (UNINF) or AD169-infected (ADI69) fibroblasts. After 16 hr of co-culture, the PBMCs were harvested, washed, and used as effector cells at an effector to target cell ratio of 50:1, against uninfected (UNINF) or AD169-infected (ADI 69) fibroblast target cells in a 4 hr cytotoxicity assay. The results are expressed as the percentage (%) lysis, and represent the mean ± standard error of triplicate values.

3.2.7 The activation of NK cel is by fibroblasts infected with CMV for various times

The results in Sections 3.2.2 and 3.2.5 showed that in a 20 hr assay, the killing of fibroblasts infected with CMV strain AD169 depended on the expression of CMV late genes. In addition, it was subsequently shown that the 20 hr assay period could be separated into 2 phases, involving the initial activation of NK cells by CMV- infected cells, followed by target cell lysis. Next, the ability of CMV-infected fibroblasts at various stages post infection, to activate NK cells was investigated. In order to examine the requirements for the activation of NK cells, PBMCs were co- cultured overnight in a transwell system with fibroblasts at various times post infection with CMV at an MOI of 2. The PBMCs were placed in the upper chamber of a transwell system, separated by a semi-porous membrane from the lower chamber, which contained the fibroblasts. After overnight co-culture, the PBMCs were harvested, washed and tested as effector cells in a 4 hr cytotoxicity assay at an effector to target cell ratio of 50:1, for their ability to lyse fibroblast target cells that had been infected with CMV for 4 days.

As shown in Figure 3-10, only fibroblasts that had been infected with CMV for at least 3 days were capable of activating NK cells such that they could specifically lyse the target cells. Once NK cells were activated, equivalent lysis was seen with infected (Figure 3-1 OB) and uninfected (Figure 3-1OA) target cells. Uninfected fibroblasts or fibroblasts infected with CMV for only 1 or 2 days failed to activate NK cells. Thus, the full activation of NK cells by co-culture with infected cells required that the cells should be infected for at least 3 to 4 days. Based on these results, fibroblasts at 4 days post infection were used to activate NK cells in all future experiments.

3.2.8 The effect of ganciclovir on the ability o f CMV-infected fibroblasts to activate NK celis

The results in Section 3.2.5 showed that in the 20 hr cytotoxicity assay, ganciclovir treatment abrogated the NK cell-mediated lysis of infected target cells, indicating that CMV late gene expression was required. Next, the question of whether CMV late gene expression was required for the activation phase or the effector phase was examined. The results above showed that fibroblasts infected with CMV could activate NK cells only at late times post infection, therefore, the effect of ganciclovir treatment of infected fibroblasts on their ability to activate NK cells was examined. NK cells were co-cultured with fibroblasts at various stages post infection with CMV, using the transwell system, as described in Section 3.2.7. The fibroblasts in the lower chamber of the transwell system were seeded on 5 consecutive days, and then either left uninfected, or infected with the AD169 strain of CMV at an MOI of 2. Immediately after infection, the cells were treated with medium alone, or with medium containing 50 pg/ml of ganciclovir. On the fifth day, PBMCs were added to the upper chamber of the transwell and incubated overnight. The PBMCs were then harvested, washed and used as effector cells at an effector to target cell ratio of 50:1, in a 4 hr cytotoxicity assay. The target cells were fibroblasts that were uninfected, or had been infected with the ADI 69 strain of CMV at an MOI of 2, for 4 days.

The results are shown in Figure 3-10. As described in the previous section, NK cells that had been co-cultured with uninfected fibroblasts were not activated and were unable to lyse the target cells. On the other hand, the co-culture of NK cells with fibroblasts that had been infected for at least 3 days resulted in their activation, such that they were capable of lysing both infected and uninfected fibroblasts. However, the ganciclovir treatment of infected cells completely inhibited the activation of NK cells, as assessed by their inability to lyse either uninfected (Figure 3-1OA) or infected (Figure 3-1 OB) fibroblasts. These data indicated that the expression of CMV late genes was necessary for the activation of NK cells by CMV- infected fibroblasts.

LYSIS OF UNINFECTED TARGET CELLS 50- 4 0 - (/) W 3 0 - 20- 10- UNINF ADI 69 AD169+GCV B

DAY 1-2 DAY 2-3 DAY 3-4 DAY 4-5 DAY 5-6